Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

Abstract

Background Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. Results A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. Conclusion We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

Synthetic Gene-Based Heterologous Expression, Proteolytic, and Structural Characterization of Caseinolytic Protease of Lactobacillus plantarum IIA-1A5

“…In this study, we used the pET28a (+) (Novagen) as the expression vector. The pET expression system is based on the T7 promoter of T7 bacteriophage that is common for recombinant proteins expression (Fiege & Dinkel, 2020).…”

On the evolution of the plant phytochrome chromophore biosynthesis

“…GST-tagged PcyA and GST-tagged mHY2 were produced and purified as described previously (Kohchi et al, 2001; Frankenberg and Lagarias, 2003). Apo-Cph1 and apo-BphP were produced as described elsewhere (Tasler et al, 2005; Fiege and Frankenberg-Dinkel, 2020).…”

Reprogramming T7RNA Polymerase in Escherichia coli Nissle 1917 under Specific Lac Operon for Efficient p-Coumaric Acid Production

“…coli strains that have been studied and catalogued, Nissle 1917 strain (EcN in short) is the only strain applied as an active pharmaceutical ingredient for targeting intestinal diseases over 100 years . Considering the genetic accessibility and modifiability, EcN was an ideal candidate to produce recombinant cytokines and antigens as well as to prevent and treat intestinal inflammation through episomal expression. − Nevertheless, only two reports investigated the T7 system in EcN for recombinant heme-dependent proteins till date, , implying that reprogramming T7RNAP in the non-model strain is still challenging.…”

“…25 Considering the genetic accessibility and modifiability, EcN was an ideal candidate to produce recombinant cytokines and antigens as well as to prevent and treat intestinal inflammation through episomal expression. 25−28 Nevertheless, only two reports investigated the T7 system in EcN for recombinant hemedependent proteins till date, 29,30 implying that reprogramming T7RNAP in the non-model strain is still challenging.…”